At the time of egg laying, the epiblast cells already are strongly polarized. They form apically localized adherens and tight junctions and express several basal membrane components such as fibronectin, laminin, and most likely several integrins in their basal membranes (Sanders, 1982; Zagris, 2001). Attached to this sheet of epithelial cells are small groups of rounded cells that form the primary hypoblast; these hypoblast cells derive from the epiblast via an ingression process known as polyingression (Eyal-Giladi and Kochav, 1976; Weinberger and Brick, 1982a). The movements associated with gastrulation begin 4 to 5 h after incubation of fertilized eggs at 37 °C. The first observable movements are associated with the formation of the secondary hypoblast, which forms in a posterior to anterior direction [Fig. ​[Fig.1A].1A]. Some of the cells that form the secondary hypoblast appear to derive from cells of Koller’s Sickle, which are moving forward (Eyal-Giladi et al., 1992; Spratt and Haas, 1960). During hypoblast development these cells flatten and fuse to form an epithelial sheet of cells, which during its forward extension also incorporates cells from the primary hypoblast (Low and McClugage, 1993). During the early stages of development there is considerable cell division both in the epiblast and in the hypoblast certainly contributing to the growth and expansion of the embryo [Fig ​[Fig1B].1B]. Evidence has, however, been presented claiming that the hypoblast can form in the absence of significant cell division, suggesting that cell movement and possibly ingression from the epiblast may be sufficient to account for hypoblast formation (Weinberger and Brick, 1982b).

The large scale cell flows that have been described for both the hypoblast and epiblast are relative to the outside world. There is little doubt that some of the observed flows must have their basis in active cell motility, but at the outset it is not clear whether all the observed flows are the result of active individual cell movements or whether some cell movements are passive displacements, possibly largely the result of changes in tissue geometry elsewhere in the embryo. Until now three distinct mechanisms had been proposed to drive streak formation (Chuai and Weijer, 2007; Wei and Mikawa, 2000):

There is abundant evidence that cells can modulate the extracellular matrix at least on a small scale by localized remodeling, for instance to accommodate growth of the embryo. This inevitably will involve making and breaking interactions between various matrix components. It will, however, be important to unequivocally demonstrate whether the extracellular matrix moves on a large scale and that the antibody label technique does accurately reflect the real dynamics of the matrix. Fibronectin after synthesis and secretion is known to exist in several distinct states, a soluble state, a cell bound state, and a state of incorporation into large fibrilar complexes (Mao and Schwarzbauer, 2005; Schwarzbauer and Sechler, 1999). Epiblast and hypoblast cells secrete soluble fibronectin, which is thought be in a globular form. It is believed that secreted fibronectin binds to α₅β₁ integrins, specific transmembrane cell surface receptors with an extracellular fibronectin binding domain. Fibronectin binding results in integrin activation, clustering, and activation of the actin cytoskeleton, which somehow unfolds the globular fibronectin molecule, allowing it to bind to and interact with other fibronectin molecules to form fibrils, which in turn interact with many other extracellular matrix components (Daley et al., 2008; Mao and Schwarzbauer, 2005). The epitope binding sites of the antibodies used in this study have not been mapped, but the antibodies used were divalent and therefore have the ability to cross link fibronectin molecules, possibly leading to a failure of suitable processing and assembly of fibronectin into functional fibrils. It seems possible that the labeling techniques used somehow immobilized fibronectin to the cell surface, resulting in cells apparently taking some of the matrix with them. This caveat could be addressed by repeating the experiments with monovalent Fab antibody fragments, which can still bind fibronectin but have lost their ability to cross link fibronectin molecules. Alternatively it would be possible to inject fluorescently labeled fibronectin, which will then become incorporated into the fibrillar network by the action of the cells as has been shown in several systems most prominently in extracellular matrix remodeling during lung morphogenesis (Larsen et al., 2006). Another way to answer this question would be to express GFP tagged fibronectin in epiblast cells and determine whether the fibronectin synthesized by cells stays with the cells or whether it is left behind in a trail as the cells move (Ohashi et al., 2002). Furthermore, it would be desirable to see whether other important protein components of the basal lamina such as laminin and especially collagen IV show the similar behavior as fibronectin, i.e., move along with the cells.

In case further experiments confirm that the matrix indeed undergoes large scale movements with similar dynamics as the cells, then there will be many questions that need to be resolved. Major questions are do the cells move actively and take the matrix with them, or is the matrix being deformed, possibly by cells elsewhere in the embryo, and does the matrix carry the epiblast cells with it, i.e., is epiblast cell displacement passive? It could be that the cells take their own matrix with them and that the main role of the matrix is signaling, for instance to stabilize apical basal polarity or at best to locally coordinate cell behaviors, but is not required for individual cells to obtain traction for active movement. If the cells move active and for some as yet unknown reason take their matrix with them, then cells would have to gain traction from other cells through cell-cell contacts. Cells could rearrange relative to other cells through active modulation of the actin filamentous network associated with adherens junctions [Fig. ​[Fig.2B].2B]. Studies in Drosophila have suggested that the cell movements associated with germband extension are based on a cell-cell intercalation mechanism that depends on local contraction of the cell-cell junction associated actin network in a myosin II dependent process (Bertet et al., 2004; Blankenship et al., 2006). This contraction is then followed by relaxation of the junctions in a direction perpendicular to the direction of the original contraction. If these contractions occur in a coordinated manner in neighboring cells, then this can result in local or large scale tissue deformations [Fig. ​[Fig.2C].2C]. Alternatively, the cells could polarize and use cellular protrusions to get traction from each other as has been suggested to occur during intercalation of mesoderm cells during convergent extension in Xenopus (Keller, 2005). Studies in Xenopus have suggested that activation of α₅β₁ integrins signaling through binding of the fibronectin of the basal lamina may suppress protrusive activity at the level of the basal lamina and thereby prevent cells moving on top of each other, thus maintaining an epithelial structure. Inhibition of integrin function through application of function blocking antibodies that prevent the binding to fibronectin result in excessive protrusive activity and loss of stability of the epithelial sheet (Davidson et al., 2008, 2006). In Xenopus it has recently been shown that inhibition of fibronectin fibril assembly by overexpression of an N terminal 70 K fibronectin domain involved in fibril formation results in randomization of the cell division planes and thereby results in a thickening of the animal cap, which prevents the radial intercalation that occurs normally, which results in a failure of epiboly and therefore proper closure of the blastoporal lip (Rozario et al., 2008). These experiments suggest that ligand dependent integrin activation is an important signaling event controlling protrusive activity and orientation of the cell division planes. Thus contact with the basal lamina is thought to be important maintaining the polarized state of the epithelium, and, in the case of the chick epiblast could play an important role in stabilizing the epithelial structure of the epiblast, functions which would be difficult to reconcile with the idea that the cells take their matrix with them. In the chick embryo both laminin and fibronectin are expressed by epiblast cells in the freshly laid egg. It has been described that blocking laminin with function blocking antibodies results in changed movements and a failure of normal streak formation (Zagris and Chung, 1990). Injection of fibronectin function blocking antibodies has been described to result in the ingression of epiblast cells into the streak, but a failure of mesenchymal cells to move away from the streak, suggesting that these cells need fibronectin to get traction to move (Harrisson et al., 1993). To assess the role of fibronectin in gastrulation, detailed loss of function experiments, i.e., deletion of fibronectin, and other extracellular matrix components are needed to assess the role of the cell matrix interactions in cell movement during streak formation.